Hermann Esterbauer, Ph.D. is a professor of biochemistry and the Head of
the Institute of Biochemistry of the Karl-Franzens-University of Graz in
Austria. He serves on the Editorial Boards of Free Radical Research Communications,
Biochemical Journal, Amino Acids, Free Radical Biology and Medicine, and
Journal of Biotechnology.

Dr. Esterbauer's major fields of research are free radical reactions and
lipid peroxidation in health and disease, particularly atherosclerosis,
and the roles of antioxidants in health and in preventing disease.

The interest in the role of vitamin E and carotenoids in the reduction of
heart disease by their preventing oxidation of low density lipoproteins
continues to grow. This is virtually the title the ground-breaking report
in the Annals of the New York Academy of Sciences in 1989 by Dr. Hermann
Esterbauer.[1] Many scientists are now following up on this relationship.
I have been interviewing the major scientists involved in this important
research over the past several months. I covered the background of vitamin
E and free radicals, but now I will begin to tie everything together in
the next two interviews.

This month I have the pleasure of bringing Dr. Esterbauer's research to
your attention, and next month, the research that started it all, that of
Dr. Daniel Steinberg's group.

Passwater: Dr. Esterbauer, when did you become interested in vitamin
E research?

Esterbauer: A long time ago. In the early 1960s I did my Ph.D. thesis
on autoxidation of fatty acids contained in plant oils. Naturally, this
evoked my interest in vitamin E as an antioxidative factor preventing rancidity.
Later on, when we studied lipid peroxidation induced by xenobiotics in liver
and liver cells, we realized, as many other investigators did, that vitamin
E is perhaps one of the most important components in cell membranes protecting
membrane lipids against oxidative damage by free radicals.

Passwater: What attracted your interest to this field of research?

Esterbauer: The University of Graz has a long tradition in lipid
and lipoprotein research, and Prof. Erwin Schauenstein, the supervisor of
my Ph.D. thesis, had the idea that perhaps some of the lipid oxidation products
formed endogenously or ingested with food have a biological or pathological
importance. More than 30 years ago, I isolated compounds from oxidized linoleic
acid. Amongst many other substances I found 4-hydroxynonenal (HNE), a substance
which is now, 30 years later, investigated in many laboratories throughout
the world as marker of oxidative stress, and as a second "toxic messenger"
of free radical damage.

Passwater: In 1987, you reported on the relationship between vitamin
E and the oxidation of the cholesterol carrier, low-density lipoprotein
(LDL). What piqued your curiosity to look at this possible relationship?

Esterbauer: In the early 1980s, the groups of Dr. Daniel Steinberg
in La Jolla and Dr. Guy Chisolm in Cleveland published some remarkable papers
on implications of the oxidation of LDL in atherosclerosis. [2-5] We were
interested in whether we could identify some substances in oxidized LDL
which we had isolated a long time ago from oxidized linoleic acid.

Together with my colleague, Dr. Gunther Jurgens, of the Institute of Medical
Biochemistry, who had worked on lipoproteins for years, we set up experiments
to oxidize LDL in vitro. Much to my surprise, the LDL, although containing
a high content of linoleic acid, was very resistant to oxidation. In an
article, which we published in 1987 in the Journal of Lipid Research, we
commented, "For one of us (H.E.), who has in the past studied lipid
peroxidation in many biological systems, the most surprising result was
the high resistance of the polyunsaturated fatty acids in LDL against oxidation."
[6]

We learned from these studies that nature protects LDL with vitamin E, carotenoids
and perhaps other not yet identified antioxidants.

Passwater: We have discussed LDL in many of my columns and interviews
over the past few years, but lipoproteins are still new subjects to the
general public. Since the understanding of how vitamin E protects against
heart disease requires a rudimentary knowledge of the role of lipoproteins,
I am still looking for help in describing them to my readers. You describe
have an excellent description. So let me ask you, what are low density lipoproteins?

Esterbauer: Cholesterol is a lipid, i. e., a fat-soluble compound.
Therefore, cholesterol is not soluble in blood, which is a water-based fluid.
To overcome this incompatibility, the body has designed a means to transport
this fat-soluble compound inside water compatible particles called lipoproteins.
There are several lipoproteins, but the two with which we are most interested
are the low density lipoproteins (LDL and the high-density lipoproteins
(HDL). In lay terms, LDL is associated with "bad" cholesterol
and HDL is associated with "good" cholesterol.

LDL primarily carries cholesterol from where it is manufactured in the liver
to various cells that need cholesterol. HDL primarily carries excess cholesterol
back to the liver. LDL is the main carrier of cholesterol in our blood stream.
In persons having normal cholesterol and other blood fats, typically about
60% of the total blood cholesterol is contained in LDL. Many epidemiological
studies and case control studies have shown that increased levels of LDL
are associated with an increased risk of atherosclerosis.

LDL is a very large spherical particle with a molecular weight of about
2.5 million, consisting of an oily central core of about l,600 molecules
of esterified cholesterol and several hundred molecules of triglycerides.
This core is surrounded by a shell of phospholipids, the polar head groups
of these molecules face the outside and make the particle soluble in blood
despite the high cholesterol and fat content.

It is important to keep in mind that LDL is not only rich in cholesterol
but also in polyunsaturated fatty acids, mainly linoleic acid, arachidonic
acid, which are - if not protected - highly susceptible to oxidation. On
average, about l.500 molecules of PUFAs are present in an LDL particle.
They clearly need protection by antioxidants. The major ones are vitamin
E and carotenoids. Vitamin E is contained in the shell, whereas B- carotene
is in the core.

Passwater: LDL is a carrier of cholesterol, but the cholesterol has
to get inside of the cell to be used. Tell us a little about the receptors
that recognize LDL, latch on to it and bring the contents into the cell
interior.

Esterbauer: Embedded in the LDL shell is also a large protein termed
apolipoprotein B. The Nobel Price winners, Dr. Joseph Goldstein and Dr.
Michael Brown, discovered that a specific receptor (termed LDL-receptor)
that can recognize apolipoprotein B of LDL is present at the surface of
most cells in our body. When LDL binds to such receptors it is quickly taken
up by the cell and the LDL particle is degraded intracellularly into its
constituents. Most of them are reused again as building blocks for membranes
and new lipoproteins.

The liver is particularly effective in removing LDL from the circulation.
On average, an LDL particle circulates in the blood for about 2 days before
it is cleared by this receptor-mediated uptake.

Passwater: For years, many researchers thought that LDL was the main
culprit in initiating the cholesterol deposits in arteries. You mentioned
that your attention was aroused by Dr. Daniel Steinberg's group's discovery
that changed the direction of heart disease. They found that it is not normal
LDL that is the problem, but oxidized-LDL, i. e., LDL that has been altered
by free radical attack or reaction with oxygen. How does oxidized-LDL differ
from normal LDL?

Esterbauer: I would like to have X-ray eyes and be able to actually
see an oxidized LDL particle. From the chemical analyses which we made,
it seems clear that it must look ugly, the beautiful architecture of normal
LDL no longer exists. The antioxidants are destroyed, the polyunsaturated
fatty acids and even the cholesterol moiety are heavily oxidized and partly
polymerized. A large number of smaller and highly reactive break-down products
segregate from the oxidizing lipids and emanate from the particle.

As recently shown by Dr. Edwin Frankel from the University of California,
Davis, some of these new products are even volatile and can be detected
in the gas phase above solutions of LDL. [7,8] Pathologically, perhaps the
most important change in oxidized-LDL is that its protein, the apolipoprotein
B, is damaged and altered to such an extent that is now binds to a "scavenger"
receptor present on the surface of specialized white blood cells called
macrophages.

Passwater: OK, now we are getting to the crux of the issue. Oxidized-LDL
is taken up by special white blood cells and make these cells look foamy.
What are these "foam cells" and what is their relationship to
atherosclerosis?

Esterbauer: Pathological, microscopic and histochemical studies have
shown that the fatty streak and plaques which form in the intima region
of the major arteries are mainly made up of cells so altered in their appearance
by engulfed LDL cholesterol that they are known as foam cells. Most of these
foam cells develop from macrophages, which again stem from more general-purpose
white blood cells called monocytes. The monocytes immigrate from the circulating
blood into the arterial wall.

For a long time it was an absolute mystery how the macrophages engulf so
much cholesterol. If macrophages were fed with normal LDL, even in high
concentration, they did not become overloaded with cholesterol, nor did
they develop to foam cells. A milestone was the discovery published by Dr.
Daniel Steinberg's group in 1984 that macrophages fed with oxidized-LDL
avidly took up this material and develop to foam cells. The uptake of oxidized
LDL occurs in an uncontrolled manner through the macrophage scavenger receptor.

Passwater: Aha, you have just provided the perfect introduction to
next month's interview with Dr. Daniel Steinberg. Dr. Steinberg will share
with us the "eureka" moments that lead to the discovery of how
antioxidant nutrients help protect us against heart disease. It is a very
interesting story. But, I still wish to develop a basic overview of this
process so that we can better understand what you have elucidated about
the interactions of the various antioxidant nutrients in preventing LDL
from oxidizing. Tell us more about the consequences of the different activity
of oxidized-LDL compared to normal LDL. How do foam cells enter the arterial
wall?

Esterbauer: Foam cells develop in an interior layer of the artery
called the intima. It is important to realize that the foam cells develop
in the arterial intima itself from resident macrophages. Foam cells do not
form in the bloodstream as an immigration of foam cells from the circulation
into the arterial wall is not possible. On the contrary, there is some evidence
that foam cells have the capacity to emigrate from the arterial intima into
the bloodstream.

Passwater: You point out that foam cells accumulate in the arteries.
The proponents of the old "cholesterol theory," based on solely
blood cholesterol levels, could not provide good reasons why cholesterol
deposits did not form in veins as well as arteries. After all, the cholesterol
concentration is the same in both arteries and veins. They attempted to
dance around that issue with various explanations, but like the cholesterol
theory itself, the answers did not stand up to scientific investigation.
Why do foam cells accumulate in arteries and not in veins?

Esterbauer: The main reason has to do with the difference in pressure
of the circulating blood in each. The lower blood pressure in veins causes
less LDL infiltration into vein walls, than the higher pressure in arteries
cause LDL infiltration into artery walls. Also, monocytes adhere to vein
surfaces (endothelium) less than artery surfaces. Therefore, foam cells
accumulate in arteries and not veins because arteries have more monocytes
adhering on the artery surfaces and because the higher blood pressure causes
infiltration of LDL.

The present opinion is that normal LDL which is continuously infiltrated
into the intima layer of arteries encounter there an "oxidative stress"
This oxidative stress is most likely mediated by activated macrophages which
have been recruited at endothelial cells at the sites of injury to the lining
of the artery.

Many other studies not directly related to atherosclerosis have shown a
remarkable feature of macrophages. If they become activated they respond
with an oxidative burst, whereby large amounts of oxygen radicals are formed.
These free radicals likely deplete the nearby environment from all water-soluble
antioxidants, as for example, vitamin C. LDL entrapped in such oxidizing
"fire" would then also be rapidly attacked and oxidized. Once
initial deposits of oxidized-LDL are formed in the arterial intima, a self-sustaining
and accelerating process can commence, since compounds released from oxidized-LDL
stimulate immigration of more and more monocyte-macrophages from the blood
to the site, where oxidized LDL is deposed.

Passwater: Now that's a disastrous chain reaction. Please summarize
what you have learned about the relationship between the antioxidant nutrients
such as vitamin E and the carotenoids so far.

Esterbauer: I can only refer to our studies on the protection of
LDL by antioxidants. One can isolate LDL from the blood and determine its
oxidation resistance. One will always observe that LDL is only oxidized
when it has lost its antioxidants. The first defense line is alpha-tocopherol
and gamma-tocopherol (vitamin E), and the last defense line is the carotenoids,
mainly beta-carotene. Much better than I can do it, Brown and Goldstein
described the situation with the words "if LDL is depleted from its
antioxidants, it is left to the mercy of oxygen" [9]

We could show by an ex vivo study that oral intake of vitamin E at daily
doses of 150, 225, 800 and 1200 IU increased the vitamin E content in LDL
in a dose dependent manner by about 40 to 110% above baseline values and
the oxidation resistance of LDL increased more or less proportionally. [10]
We also learned that the antioxidant efficacy of vitamin E varies rather
strongly between individuals, the reason for that is still not known. With
beta-carotene the situation is even more complex, in some subjects, as for
example vegetarians or patients with fat-malabsorption, we found that oral
intake of beta-carotene can significantly increase the oxidation resistance
of LDL. But in other healthy subjects beta-carotene supplementation for
3 weeks had no effect whatsoever.

For adults the RDA for vitamin E is 15 IU per day, and I think this is too
low to provide an adequate protection for LDL. One should also consider
that very strong individual variations exist in absorption of vitamin E
and its incorporation into LDL. Furthermore, the oxidative stress situation
of individuals is variable.

So far, no consensus exists on the optimal dose of vitamin E. Professor
Fred Gey from the University of Bern (Switzerland) recommends a plasma vitamin
E level of around 30 micro molar. Such a level can perhaps be reached by
most persons with an intake of about 100-200 IU per day.

Finally, I want to say that we must not only think of vitamin E but also
on all other antioxidant nutrients, such as vitamin C, beta-carotene, selenium
and perhaps others, such as flavonoids. In our body these antioxidants usually
act in a concerted and synergistic way and only an optimal balance of all
of them will ensure, at least in my opinion, an optimal health.

Passwater: Several scientists that I have interviewed in this series
have mentioned that they have followed your lead in researching the role
of antioxidant nutrients in protection against heart disease. What additional
information have they added?

Esterbauer: This is a very kind comment by them. During this interview
I have already mentioned several prominent scientists who contributed much
more to the LDL oxidation theory of atherosclerosis than I did. Our major
contribution, perhaps, was that we introduced quantitative clinical-chemical
assays, which enable us and others to measure oxidation resistance of LDL
and the protective effect of antioxidants. We have now so many biochemical
and epidemiological evidence in support of the oxidation theory, what we
need is a support by experimental animal studies, clinical studies and intervention
trials. I want to mention in this context the work by Dr. Anthony Verlangieri
from the University of Mississippi, who showed that in primates vitamin
E is prophylactically and therapeutically effective in atherosclerosis.
[11,12]

Dr. Jan Regnstrom from the Karolinska Institute in Stockholm studied the
oxidation resistance of LDL in survivors of myocardial infarction and found
a significant inverse correlation between severity of coronary atherosclerosis
and oxidation resistance of LDL. [13]

Finally, I want to address again the important work by Professor Daniel
Steinberg from the University of California, San Diego. He organized and
chaired a round table consensus conference on antioxidants in prevention
of human atherosclerosis, which was supported by the National Heart, Lung
and Blood Institute in September 1991 in Bethesda, MD. The summary of the
Meeting was published, and I quote from this paper, "It was the consensus
that the evidence available justify a clinical trial of natural antioxidants."
[14]

Passwater: What will you be investigating about this relationship
next?

Esterbauer: As I mentioned earlier, the efficacy of vitamin E to
protect LDL against oxidation varies strongly between individuals, and we
investigate presently the underlying reasons. Perhaps, genetic factors play
an important role besides dietary factors. Another project of my group deals
with the development of ELISAs (enzyme linked immunosorbent assay) for measuring
oxidized LDL and other proteins damaged by oxygen radicals and lipid peroxidation
in plasma, tissue and single cells. As you know, many researchers believe
that oxidative stress is a major cause of many diseases. If this is so,
assays to measure oxidatively damaged proteins should have a prognostic
and diagnostic value.

Passwater: Thank you Professor Esterbauer for your lucid explanations
in explaining your research to us.

Richard A. Passwater, Ph.D. has been a research biochemist since 1959. His first areas of research was in the development of pharmaceuticals and analytical chemistry. His laboratory research led to his discovery of......more

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